Derivatives of alpha-aminobenzylpenicillin



,thereof.

3,489,746 DERIVATIVES OF a-AMINOBENZYLPENICILLIN Leonard Bruce Crast, In, North Syracuse, N.Y., assignor to Bristol-Myers Company, New York, N.Y., a corporation of Delaware No Drawing. Filed Sept. 5, 1967, Ser. No. 665,291 Int. Cl. C07d 99/20, 99/16; A61k 21/00 US. Cl. 260239.1 8 Claims ABSTRACT OF DISCLOSURE 6-[D a a amino tit-(3-chloro-4-hydroxyphenyl)- acetamido1penicillanic acid and 6-[D-()-2,2-dimethyl-4- (3 chloro 4 hydroxyphenyl) 5-oxo-1-imidazolidinyl] penicillanic acid and the salts thereof are new synthetic compounds of value as antibacterial agents and in the treatment of bacterial infections. 1

BACKGROUND OF THE INVENTION Field of the invention This invention relates to novel synthetic compounds of value as antibacterial agents, as nutritional supplements in animal feeds, as agents for the treatment of mastitis in cattle and as therapeutic agents in poultry and animals, including man, in the treatment of infectious diseases caused by gram-positive and gram-negative bacteria.

Description of the prior art t SUMMARY OF THE INVENTION The compounds of this invention are 6-[D-()-a amino a (3-chloro-4-hydroxyphenol)-acetamido]pencillanic acid having the formula and 6 [D 2,2-dimethyl-4-(3-chloro-4-hydroxyphenyl)-5-oxo-1-imidazolidinyl]penicillanic acid having and the nontoxic pharmaceutically acceptable salts The nontoxic, pharmaceutically acceptable salts include for example, 1) nontoxic pharmaceutically acceptable salts of the acidic carboxylic acid group such as the sodium, potassium, calcium, aluminum and ammonium salts "and nontoxic substituted ammonium salts with amines such as tri(lower)alkylamines, procaine, dibenzylamine, N- 'benzyl-beta-phenethylamine, l-ephenamine, N,N-diben- Zylethyle'nediamine, dehydroabietylamine, N,N'-dehydro- United States Patent abietylethylenediamine, N-(lower)alkylpiperidines, such as N-ethylpiperidine and other amines which have been used to form salts of benzylpenicillin; and (2) nontoxic pharmaceutically acceptable acid addition salts (i.e. salts of the basic nitrogen) such as (a) the mineral acid addition such as the hydrochloride, hydrobromide, hydroiodide, sulfate, sulfamate, sulfonate, phosphate, etc. and (b) the organic acid addition salts such as the maleate, acetate, citrate, tartrate, oxalate, succinate, benzoate, fumarate, malate, mandelate, ascorbate, fi-naphthalene sulfonate, p-toluenesulfonate and the like. Also included are the easily hydrolyzed esters or amides of such acids which may be converted to the free acid form by chemical or enzymatic hydrolysis.

The compounds of Formula I of the present invention are prepared in the form in which the u-amino group is blocked by the reaction of 6-aminopenicillanic acid (preferably in the form of a neutral salt such as the sodium salt or the triethylamine salt) with a mixed anhydride e.g. mixed anhydride obtained from the reaction with ethyl chlorocarbonate, of an acid having the formula or the formula or with its functional equivalent as an acylating agent for a primary amino group. Such mixed anhydrides include particularly the mixed anhydrides prepared from stronger acids such as the lower aliphatic monoesters of carbonic acid, of alkyl and aryl sulfonic acids and of more hindered acids such as diphenylacetic acid. Such equivalents include the corresponding carboxylic chlorides, bromides, and then acid anhydrides. In addition, an acid azide or an active ester or thioester (e.g. with p-nitro'phenol, 2,4- dinitrophenol, thiophenol, thioacetic acid) may be used or the free acid itself may be coupled with 6-aminopencillanic acid after first reacting said free acid with N,N'-di'methylchloroformiminium chloride [cf. Great Britain 1,008,170 and Novak and Weichet, ExperientiaXXI/ 6, 360 (1965)] or by the use of enzymes or of an N,N'-carbonyldiimidazole or an N,N'- carbonylditriazole [cf. South African Patent Specification 63/2684] of a carbodiimide reagent [especially N,N'-dicyclohexylcarbodiimide, N,N'-dissopropylcarbodiimide or N-cyclohexyl-N-(Z-morpholino- 3 Soc. 83, 1010 (1961)]. Another equivalent of the acid chloride is a corresponding azolide, i.e. an amide of the corresponding acid whose amide nitrogen is a member of a quasiaromatic five-membered ring containing at least two nitrogen atoms, i.e. imidazole, pyrazole, the triazoles,

benzimidazole, benzotriazole and their substituted derivatives. As an example of the general method for the preparation of an azolide, N,N-carbonyldiimidazole is reacted with a carboxylic acid in equimolar proportions at room temperature in tetrahydrofuran, chloroform, dimethylformamide or a similar inert solvent to form the carboxylic acid imidazolide in practically quantitative yield with liberation of carbon dioxide and one mole of imidazole.

ucts of the present invention, e.g. the Z-hydroxy-lnaphth-carbonyl group is removed by acid hydrolysis, the

carbobenzyloxy group is removed by catalytic hydrogena- NHa III.

as cam-Q-on-conr Nmon Hog Kidney Acylase Thus the present invention. includes the process of preparing the compound of the formula which comprises. mixing a penicillin of Formula 'I with at least an equimolar weight ofacetone in the absence of substantial amounts of water at a pH in therange of, 5 to 9 and at a temperature in the range of 20 C. to C. D-()-2-(p-hydroxyphenyl)-glycine used as a starting material for the preparation of the compounds of this invention is prepared according to the following reaction scheme.

n omoQ-o-uz 37 0. H01 [CHaO-Q-(E H-CN] T oH30cH-con1 ing penicillin of Formula I.

Although some reaction will occur no matter what molar proportion of reactants is used, it is preferable in order to obtain maximum yields to use a molar excess of the acetone and the latter may Well be used as the reaction solvent. Water is split off during the reaction and it is thus preferable not to have a major amount of water in the reaction medium. The pH of the reaction mixture should be from about 5 to 9 and preferably on the alkaline side. The pH may be adjusted to within this range, if

necessary, by the addition of an alkaline material such as, for example, sodium hydroxide, sodium carbonate,

potassium hydroxide, potassium carbonate, ammonium hydroxide, ammonium carbonate, organic amines (e.g. triethylamine), etc.

The temperature during the reaction is not critical.

The reaction .will proceed satisfactorily at room temperature and may be-hastened by heating.

NH: NHz

To a stirred solution of 19.6 g. (0.4 mole) of NaCN in ml. of H 0 was added 23.6 g. (0.450 mole) of NH CI and 20 ml. of cone. NH OH followed by 54.5 g. (0.4 mole) of anisaldehyde in 160 ml. of methanol and the temperature maintained at 37 C. for two hours. The methanol was then removed in vacuo and the remaining mixture extracted with two ml. portions of methyl isobutyl ketone (MIBK) and combined. The combined MIB K extracts were washed once with 30 ml. of H 0 and then 240 ml. of 6 NHCI added with good mixing and the MIBK was removed in vacuo. The resulting slurry was heated at reflux (now in solution) for two hours. One hundred ml. of H 0 was added to the hot solution and then 8 g. of decolorizing carbon added and after ten minutes at gentle reflux the carbon wasfiltered off and .5 After one hour the product was filtered off, washed with 3x100 ml. H and air dried. Yield 40 g.; dec. 244 C.

with sublimation at 230 C.

II. dl-2-(p-methoxyphenyl)-N-(ch1oroacetyl) glycine To a stirred suspension of 36g. (0.2 mole) of dl-2- (n-niethoxyphenyl)-glycine in 500 ml. of H 0 was added 8g. (0.2 mole) of NaOH pellets and when a clear solution was obtained the solution was cooled to C. and with vigorous stirring 68.2 g. (0.4 mole) of chloroacetic anhydride (warm) was added all at once-Then a solution of 16 g.'i(0.4 mole) of NaOH in 100 ml. of H 0 was added over a to minute period. More NaOH was added as needed to keep the pH at-about 9 for a 1.5 hour period. Next, the pH was adjusted to 2 with 40% 'H' PO The product crystallized immediately and was -filtered oiflwashed with water and recrystallized from ethanol-water to-give 38 g. of product melting at 182- -183 C. v

'Analysis.Calcd. for' C H ClNO C, 51.21; H, 4.69. Found: C, 51.49; H, 4.90..

III. 1).- -2-(p-methoxyphenyl) -N-(ch1oroacety1) glycine and L -2- p-methoxyphenyl -glycine To 800 ml. of H 0 stirred at 37 C. was added 38 g. (0.148 -mole) of dl-2-(p-methoxyphenyl)-N-(chloroace- ,tyl) glycine and NH OH added dropwise until pH 7.8 was obtained. To the resulting solution was added 2 g. of Hog Kidney Acylase (Sigma Chemical Company) and stirring continued. at 37 C. (internal) for 21 hours. The solids containing crude L-(+)-2-(p-methoxyphenyl) glycine were thenfiltered ofi and washed with 2 100 ml. H 0 and the pH of the combined filtrates adjusted to 4-5 with glacial. acetic acid. This solution was heated on the steam bath for 30min. with 5 g. of decolorizing carbon and then -filtered; The carbon cake was washed with 50 ml. of ;warm water, and the combined filtrates cooled and acidified to pH 2 with 40% H PO After one hour cooling at 0 C. the crystalline product was filtered off and washed with cold water (3 X) and air dried. The yield .was 16 g. A seeondrun using five times the. previous amounts gave a yield of 83 g. (87% yield); M.P. 170-171 C.; [oc] l93 (C=1% ethanol)."

Analysis.-+Ca lcd. for C H ClNO C, 51.21; H, 4.69. Found: C, 51.50; H, 4.99. I When thes'oli'dscontaining crude L-(+)-2-(p-methoxyphenyD-N l chloroacetylglycine are treated with hot 3 NHCl" (200 ml.) and carbon followed by filtration and pH adjustment to 5.5/there is obtained 6 g. (first run) of pure L- -2-(p-me thoxyphenyl) glycine.

25 c.'+ 5 4 (C=1%, 1 NCHl) IV, D-( -2- (p.-methoxypheny1) -glycine The 16 g. of D-()-2- (p-methoxyphenyl)-N-chloro acetylglycine Was refluxed 1.5 hours in 170 ml. of 2 NHCl. The resulting clear solution was filtered and cooledat 5 C. and the pH adjusted to 5.5 with NH OH. The product was then filtered off after cooling min. and washed with 3 x25 ml. of cold water. The dried material D-()- 2-(p-methoxyphenyl)-glycine weighed 9.5 g. A second run gave 54 g. using the 83 g. of starting material from III.

MP a -149.9 (C=1%, 1 NHCl) (first run) [a] 148.1' (C=1%, 1 NHCl) (second run) Analysis.-Calcd. for C H NO C, 59.67; H, 6.13;

N, 7.74. Found: C, 59.38; H, 6.16; N, 8.00.

V, D- -2- (p-hydroxyphenyl) -glycine A mixture of 1.81 g. (0.01 mole) of D-()-2-(pmethoxyphenyl)glycine. ([a] Q 149.9 C=1%, 1 NHCl) and 10 ml. of 48% HBr was heated at gentle refiux for 2 hours. The resulting solution was concentrated at reduced pressure at 30 C. to a wet solid. A minimum amount of water (20 C.) was added to dissolve the HBr salt and with cooling NH OH was added to pH 5.

The resulting thick gel which ppt. was warmed to 50 C. and when solution was nearly obtained a different crystalline form began to ppt. Upon cooling 30 min. at 05 C. there was obtained 990 mg. of cold water washed (3X1 ml.) and air dried material, D-(-)-2-(phydroxyphenyl) glycine.

[a] 161.2 (C=1%, 1 NHCl) dec. pt. 223 C.

A second run using 20X the above amounts gave 24.5 g. of material.

M13 -153 (C=1%, 1 NHCl) Analysis.-Calcd. for C H NO C, 57.49; H, 5.43; N, 8.39. Found: C, 57.41; H, 5.67; N, 8.39.

The compounds of the present invention are useful in the treatment of infections caused by gram-positive and gram-negative bacteria.

In addition, the compounds of the present invention are orally absorbed.

In the treatment of bacterial infections in man, the compounds of this invention are administered orally or parenterally, in accordance with conventional procedures for antibiotic administration, in an amount of from about 5 to 60 mg./kg./day and preferably about 20 mg./ kg./ day in divided dosages, e.g. three or four times a day. They are administered in dosage units containing, for example, 125 or 250 or 500 mg. of active ingredient with suitable physiologically acceptable carriers or excipients'. The dosage units can be in the form of liquid preparations such as solutions, dispersionsor emulsions or in solid form such as tablets, capsules, etc.

The following examples will serve to illustrate this invention without limiting it thereto.

EXAMPLE 1 D- -0t-8.IIlll10-0L- 3-ch1oro-4-hydroxyphenyl) glycine stirring, 250 ml. of dry ether was added slowly and crystallization began. After 15 min. the product was filtered oif, washed with dry ether and air dried. The 7 g. obtained was dissolved in 50 ml. of 1 NHCl, filtered, and the pH adjusted, with cooling to 5 with cone. NH OH. The resulting crystalline product was filtered off. after 5 min. standing, washed with two 20 ml. portions of water and 5X with acetone. The vacuum dried material weighed 4.6 g.; dec. pt. 217 C. (sharp). The NMR and IR spectra were consistent with the desired structure.

C, -137.1 (C=1%,1NHC1) Analysis.Calcd. for C H ClNO C, 47.76; H, 4.01; Cl, 17.66. Found: C, 47.16; H, 3.92; Cl, 17.96.

EXAMPLE 2 Sodium D- -N- (Z-hydroxyl-napthylmethylene) -zxamino-a- 3 -chloro-4-hydroxyphenyl -acetate To a stirred solution of 8 g. (0.04 mole) of D-()-2- (3-chloro-4-hydroxyphenyl)-glycine, 25 ml. H 0, 10 ml. ethanol, and 1.6 g. (0.04 mole) of sodium hydroxide was added, all at once, a warm solution of 7.57 g. (0.044 mole) of 2-hydroxy-l-naphthalaldehyde (Aldrich Chemical Company) in 40 ml. of 95% ethanol. The mixture was heated until an initial precipitate redissolved and then was rapidly cooled to about 5 C. and scratched. After cooling one hour in the ice bath the crystalline product was filtered 01f and air dried. The bright yellow product was recrystallized from ethanol-20% water to give 'spectra were entirely consistent with the desired structure.

Analysis.-Calcd. for C H ClNO Na: C, 60.37; H, 3.47. Found: C, 60.66; H, 3.72.

Example 3 6-[D-()-2,2 dirnethyl-4-(3-chloro-4-hydroxyphenyl)-5 oxo-2-(H)-1-imidazolidinyl]-penicillanic acid To a stirred and cooled (10 C.) suspension of 3.78 g. (0.01 mole) of sodium D-()-N-(2-hydroxy-l-naphthyl- -methylene) or. amino-u-(3-chloro-4-hydroxyphenyl)- acetate in 100 ml. of acetone, 5 ml. of p-dioxane and 3 drops of pyridine was added 1.08 g. (0.01 mole) of ethyl chloroformate (EKC). The mixture was stirred at C. for 30 minutes and then cooled to 40 C. and filtered to remove the sodium chloride which precipitated. To this filtrate of the mixed anhydride, vigorously stirred at C., was added all at once, a precooled (0 C.) solution of 2.16 g. (0.01 mole) of fi-aminopenicillanic acid, 1.68

g. (0.02 mole) of NaHCO in 50 ml. of Water. There' 30 minutes at 22 C. and then two 300 ml. ether extracts were taken and discarded. The pH was readjusted to 4.7 with NaOH and concentrated under reduced pressure to a volume of 25 ml. at 20 C. A small amount of insoluble material was filtered off and 25 ml. acetone added to the filtrate. The pH was then adjusted to 8.8 with 20% NaOH and after 3 hours the pH was adjusted to 3 with H PO and two 100 ml. ethyl acetate extracts taken. The combined ethyl acetate extracts were washed once with 20 ml. H 0 and then filtered and concentrated under reduced pressure at 15 C. to a volume of about 20 ml. The crystalline product was filtered off and slurried in 10 ml. of acetone-water (1:1 by volume) for 10 minutes and filtered again.

The yield was 280 mg. of product decomposing at 182 C. and having IR and NMR spectra entirely consistent with the proposed structure.

Analysis.Calcd. for C H ClN G C, 51.82; H, 5.04. Found: C, 48.39; H, 5.28.

This product is found to inhibit Staphylococcus aureus Smith at a concentration of 0.063 mg./ml., Streptococcus pyogenes at a concentration of 0.004 mg./ml., Staphylm coccus aureus BX-1633-2 (a strain resistant to benzylpenicillin) at a concentration of 63 mg./ml., Escherichia coli Juhl at a concentration of 2 mg./ml., Salmonella enteritidis at a concentration of 0.125 mg./ml., and Diplococcus pneumoniae at a concentration of 0.008. mg./ml.,

and to exhibit upon oral administration in mice a CD against Staph. aurezzs Smith of 0.5 mg./kg.'

EXAMPLE 4 To a stirred suspension of 600 mg. of 6-[D-()-2,2- dirnethyl 4 (3 chloro-4-hydroxyphenyl)-5-oxo 1-imidazolidinyl]- penicillanic acid in 5 ml. water. is added 20% sodium hydroxide solution until pH 7 is obtained. The pH is maintained at about 7 with occasional addition of 1 N HCl for four hours and then the pH is adjusted to 4.5 with 1 N HCl and maintained at pH 4.5 for another. hour. The crystalline precipitate that forms is separated by, filtration, washed with water and dried inyacuo over P 0 yielding 102 mg. of the product 6-[D-(-)cg-amino -al-(3- chloro 4 hydroxy phenyl)-acetamido]penicillanic acid. The infrared spectrum is consistent with the proposed structure having a peak at 1600 cmf The product is found to inhibit Stdphylococcusaureus Smith at a concentration of 0.032 mg./ml., Streptococcus pyogenes at a concentration of 0.004 mg./ml., Staphylococcus aureus BX-l6 332 (a strain resistant to benzylpenicillin) at a concentration of 63"mg./ml., Escherichia coli Juhl at a concentration of 4 mgjmlf, Salmonella: enteritidis at a concentration-of 0.125 mg./ml. and Diplococcus pneumoniae at a concentration of 0.004 mg./ml., to exhibit upon oral administrationin mice a CD against Staph. aureus Smith of 0.5 mg./kg. -f 1 1 1. 6 [D a (3-chloro-4-hydroxyphenyD-uamino-acetamido]penicillanic' acid and its nontoxic, pharmaceutically acceptable salts. i

2. The sodium salt of the compound of claim 1.

3. The potassium salt of the compound of claim 1.

4. The triethylamine salt of the compound of claim 1.

5. 6 [D 2,2-dimethyl-4-(3-chloro 4-hydroxyphenyl) 5 oxo 1 imidazolidinyl]penicillanic acid and its nontoxic pharmaceutically acceptable salts.

6. The sodium salt of the compound of claim 5.

7. The potassium salt of the compound of claim 5.

8. The triethylamine salt of the compound of claim 5.

References Cited UNITED STATES PATENTS 3,071,575 1/1963 Doyle et at". 260239.1 3,256,272 6/1966 Ito et a1. 260-2391 2,985,648 5/1961 Doyleet a1. 260 239.1

FOREIGN PATENTS 978,178 12/1964 Great Britain.

NICHOLAS S. RIZZO, Primary Examiner US. Cl. X.R. 

